The overall objective of this project has been to attempt to understand the structure-function relationships in the two forms of malate dehydrogenase from porcine heart. Recent work by this laboratory has implicated the essentiality of four residues in the active center of the mitochondrial enzyme (a histidine, a cysteine, a lysine and an arginine) by means of selective chemical modification. Similar studies in the cytoplasmic enzyme has yielded information about a lysine and an arginine residue. Sequence studies are being pursued on labeled peptides containing these residues in order to identify other residues in their proximity. More recently, we have established the existence of a pH and protein concentration dependent dissociation of the mitochondrial enzyme. Solvent perturbation studies and studies involving proton uptake and release upon binding of cofactors to native and modified enzyme will allow us to correlate the role of specific residues in this dissociation of the subunits of this enzyme. These enzymes with the detailed information we have now collected act as an excellent vehicle for attempts to understand the general mechanism of action of dehydrogenase enzymes.